In biochemical analysis, by way of example, there is a high demand for arrangements and methods by means of which, using biochemical or biological or synthetic identification elements immobilized on a surface, one or a plurality of analytes in a supplied sample are to be detected with high selectivity and sensitivity. In this case, many known detection methods are based on the determination of one or a plurality of luminescences in the presence of the one or the plurality of analytes.
In this case, the term “luminescence” in this application denotes the spontaneous emission of photons in the ultraviolet to infrared range after optical or non-optical, such as, for example, electrical or chemical or biochemical or thermal excitation. By way of example, chemiluminescence, bioluminescence, electroluminescence and, in particular, fluorescence and phosphorescence are also encompassed by the term “luminescence”.
The term “optical transparency of a material” is used hereinafter in the sense of requiring the transparency of this material at at least one excitation wavelength. At a longer or shorter wavelength, this material may also be absorbent.
By means of thin-film waveguides having a high refractive index, based on a thin wave-guiding film only a few hundred nanometers thick on a transparent carrier material, it has been possible in recent years to significantly increase the sensitivity for an analyte detection. This exploits the fact that the guiding of light in the material of a waveguide that has a high refractive index is associated with the formation of a so-called evanescent field which has a penetration depth of a molecular order of magnitude, that is to say of a few hundred nanometers, into the adjacent media having a lower refractive index. A strictly surface-bound excitation field is thus available which permits processes to be studied selectively within a layer thickness defined by the penetration depth of the evanescent field into the media having a low refractive index. By way of example, WO 95/33197 describes a method in which the excitation light is coupled into the wave-guiding film of a sensor platform via a relief grating as diffractive optical element. The surface of the sensor platform is brought into contact with a sample containing the analyte, and the isotropically emitted luminescence of substances capable of luminescence that are situated at the penetration depth of the evanescent field is measured by means of suitable measuring devices such as, for example, photodiodes, photomultipliers or CCD cameras. It is also possible for that portion of the radiation generated in evanescent fashion which is fed back into the waveguide to be coupled out and measured via a diffractive optical element, for example a grating. This method is described for example in WO 95/33198.
The terms “evanescent field” and “near field” are used synonymously below.
One disadvantage of the method for detecting luminescence excited in evanescent fashion as described previously in the prior art, in particular in WO 95/33197 and WO 95/33198, is that in each case only one sample is analyzed by means of the embodiments of sensor platforms described therein. In order to be able to carry out further measurements on the same sensor platform, complicated washing or cleaning steps are continuously required. This holds true particularly if an analyte that is different from the first measurement is intended to be detected. In the case of an immunoassay, this generally means that the entire immobilized layer on the sensor platform has to be exchanged or equally a new sensor platform as a whole has to be used.
For simultaneously or successively carrying out luminescence-based multiple measurements with essentially monomodal, planar inorganic waveguides, WO 96/35940, for example, has disclosed devices (arrays) in which at least two separate wave-guiding regions that are irradiated separately with excitation light are arranged on a sensor platform. However, dividing the sensor platform into separate wave-guiding regions disadvantageously has the consequence that the space requirement for discrete measurement regions, in discrete wave-guiding regions on the common sensor platform is relatively large and, therefore, it is again possible to achieve only a comparatively low density of different measurement zones (or so-called “features”).
Therefore, there was a need to increase the feature density or to reduce the area required per measurement region.
Based on simple glass or microscope laminae, without additional wave-guiding layers, arrays are known in the form of so-called “microarrays” with a very high feature density. By way of example, U.S. Pat. No. 5,445,934 (Affymax Technologies) describes and claims arrays of oligonucleotides having a density of more than 1000 features per square centimeter for the detection of nucleic acids with complementary (partial) sequences. The excitation and the read-out of such arrays are based on traditional optical arrangements and methods. The entire array can be illuminated simultaneously with an expanded excitation light bundle, but this leads to a relatively low sensitivity since the excitation is not restricted to the interacting surface and since, moreover, the scattered light portion is relatively large and scattered light or background fluorescent light from the glass substrate is also generated in the regions in which there are no oligonucleotides immobilized for the binding of the analyte. In order to increase the excitation intensity and to improve the sensitivity during detection, frequently confocal measuring arrangements are used and the different features are read out sequentially by means of “scanning”. However, this results in a longer expenditure of time for the read-out of a large array and a relatively complex optical construction.
Recently it was possible to show that the format of such “microarrays” can be applied to planar thin-film waveguides as carriers (sensor platform) without the portion of the luminescence which is excited in the evanescent field and is fed back into the waveguide leading to a significant crosstalk of signals from different measurement regions. With this arrangement it was possible to achieve a significant increase in the sensitivity, by a factor of 50 to 100, in comparison with the conventional measuring arrangements [G. L. Duveneck et al. Analytica Chemica Acta 469 (2002) 49-61].
For all of the configurations mentioned, however, ultimately primarily the background signals and the associated background noise remain limiting factors for the detection limits that can respectively be achieved. This is caused for these as well as for all the abovementioned excitation and detection configurations inter alia by the fact that in the case of most of the luminescent dyes used, the spectral distance between excitation and emission wavelengths (Stokes Shift) is relatively small, typically between 20 nm and 50 nm. Although some luminescent dyes are known which have a large Stokes Shift, up to approximately 300 nm, such as some lanthanide complexes, for example, these disadvantageously generally have a relatively low quantum efficiency and/or low photostability.
Moreover, in the case of the known arrangements based on thin-film waveguides having a high refractive index, for example based on wave-guiding layers made of Ta2O5 or TiO2, with conventional excitation, it is disadvantageous that the propagation losses of these waveguides and also the intrinsic fluorescence of these thin-film waveguides (for example through traces of fluorescent contaminants in the carrier layer (b)), rise drastically at short excitation wavelengths. Thus, short-wave excitation is limited here at approximately 450 nm to 500 nm. An arrangement would be desirable, however, by means of which fluorophores can be excited even at shorter wavelengths and their luminescences can be detected with a lowest possible or at best even without any background.
Methods based on multiphoton excitation, in particular two-photon excitation, have been known for some years. However, a two-photon excitation requires extremely high field strengths or intensities of the excitation light. These are achieved in the arrangements described by means of powerful pulsed lasers having extremely short pulse lengths (typically of femtoseconds). Previous systems use optical arrangements which are associated with very high system costs and make high requirements of the technical qualification of the user. Therefore, they are not suitable for more routine applications, outside the area of research. The required intensity densities have previously been achieved for example by means of pulsed high-power lasers in confocal microscopic arrangements, as is described for example in U.S. Pat. No. 5,034,613, with a laser focus diameter of less than 1 micrometer in the focal plane of the microscope. However, measuring an extended area by means of scanning again disadvantageously also requires a high expenditure of time besides the high outlay on instrumentation.
It was recently shown that with the aid of planar thin-film waveguides it is possible to carry out two-photon luminescence excitation not only in a microscopic excitation beam diameter, but microscopically on areas of several square millimeters at the surface of a suitable thin-film waveguide [Duveneck, G. L., et al. “Evanescent-field-induced two-photon fluorescence: excitation of macroscopic areas of planar waveguides; Applied Physics B, 73 (2001) 869-871; WO 01/79821; WO 02/79765]. This novel combination of waveguide technology with two-photon luminescence excitation makes it possible for the long-wave excitation light to be spectrally distinctly separated from the shorter-wave emission light. However, this advantage of the greater spectral separation is partly canceled out again by virtue of the fact that, on account of the significantly lower quantum efficiencies of the two-photon-induced luminescence, in comparison with the conventional one-photon-induced luminescence, the ratio between only very weak emission intensity and very strong intensity of the excitation scattered light is significantly less favorable than in the case of the one-photon-induced luminescence.
Therefore, there is a need for a method and an analytical measuring arrangement by means of which an effective separation from the excitation light (excitation signal) can also be carried out for the two-photon-induced luminescence, as an example of optical signals which follow the intensity of an excitation signal in a nonlinear manner. At the same time it is desirable to advantageously utilize the specific property of the nonlinear correlation with the excitation light for such a method and such a measuring arrangement.
Spectral separation methods have the general disadvantage of never completely suppressing the excitation light (excitation signal) to be discriminated and of likewise incomplete transmission for the emission wavelength to be detected. In addition, by way of example, the effectiveness of interference filters is disadvantageously also significantly dependent on the angle at which the light impinges on the filter.
An elegant alternative to the spectral separation of excitation and emission signals consists in the so-called “lock-in” technique, which has already been known for a long time for point-type signal detection (that is to say in combination with zero-dimensional detectors) [Meade, M. L., “Lock-in amplifiers: principles and applications”, London (1983), Peregrinus 232]. The basic principle of this technique is based on the modulation of the excitation signal with a selectable modulation frequency and the detection of the emission signal in a manner correlated with this modulation frequency. By way of example, the modulation of the excitation light intensity impinging on an interaction volume or on an interaction layer may be effected with the aid of a rotating disk with an arrangement of slits and closed regions (a so-called “chopper”), whereby the excitation light path is alternately blocked and released. As an alternative, it is also possible, for example, to modulate the excitation current of a laser diode. The resulting emission light under the periodically varying illumination conditions (in the absence and presence of excitation light) is directed onto a detector whose response time corresponds at least to the modulation frequency. An associated so-called lock-in amplifier amplifies only those signals which are correlated with the modulation frequency, by forming the difference between the detector signals forwarded to it at the different phases. The light portions measured by the detector which are not correlated with the modulated excitation intensity are thus eliminated. Under suitable conditions, the lock-in technique also makes it possible, by measuring the phase difference between maximum excitation light intensity and maximum emission light intensity, to determine the decay time, that is to say the life time of this emission after pulsed excitation.
In the case of the traditional lock-in technique described above, typically the signal portions which are proportional to the excitation light are selected for further processing by means of an individual detector (“point detector”) and response signal portions which are not correlated with the excitation signal are rejected.
Various work on applying the lock-in technique described or methods related thereto to one- or two-dimensional detector arrays is known. WO 96/15625 describes a device and a method for detecting an intensity-modulated radiation field, the demodulation process being integrated electronically in a detector array. This method is suitable for example for propagation time measurements (“time of flight”, TOF) or heterodyne interference measurements of remote moving objects. The method is based on the filtering of linear signal portions from the background light. The distance from an object, for example, is determined from the measured propagation time or phase shift in an interferometric method. It is mentioned in the description that, in the case of a sinusoidally modulated radiation field, for a number of four samplings (signal measurements) per period it is possible to determine the amplitude, the phase and the background light of the radiation field, and that when the sampling rate is increased, it is possible to determine further parameters of the radiation field, such as, for example, Fourier coefficients. However, no statements whatsoever are made with regard to how such a further development could be realized, rather exclusively the detection of linear signal portions of the radiation field is described. A publication [T. Spirig, P. Seitz, O. Vietze and F. Heitger, “The Lock-In CCD—Two-Dimensional Synchronous Detection of Light”, IEEE Journal of Quantum Electronics 31 (1995) 1705-1708] of the underlying work describes the technical implementation of the detector in greater detail, where it is referred to as a “Lock-In CDD” (CCD: “Charge-coupled device”). Further developments of this arrangement are described in T. Spirig, M. Marley and P. Seitz, “The Multitap Lock-In CCD with Offset Subtraction”, IEEE Transactions on Electronic Devices 44 (1997) 1643-1647 and in the International patent application WO 01/84182. However, neither work contains any indications whatsoever about possibilities for detecting signal portions correlated nonlinearly with the intensity of an excitation field available at the measurement location.
By contrast, the present invention relates to the separation of the nonlinear signal portions from the remaining portions which are linearly correlated or uncorrelated with a modulated excitation field. Following the present invention, then, the intention is for precisely those signal portions to be rejected whose detection is the object of the invention described in WO 96/15626 and the further publications mentioned above. Moreover, the arrangement according to the invention and the method according to the invention which is to be performed therewith have the advantage that they permit the use of any desired one- or two-dimensional detector array, provided that the electronic response time thereof is short enough to follow the frequency of the modulation of the excitation light power and thus the modulation of the intensity of the light that is to be detected and emerges from the measurement location. The use of detectors such as the “Lock-In CCDs” described is possible, but in no way necessary, for the arrangement according to the invention and the measuring method according to the invention.
The application of two-dimensionally spatially resolved phase-sensitive detection in interference microscopy was recently disclosed [A. Dubois, “Phase-map measurements by interferometry with sinusoidal phase modulation and four integrating buckets”, J. Opt. Soc. Am. A 18 (2001) 1972-1979; A. Dubois, L. Vabre, A. C. Boccara and E. Beaurepaire, “High-resolution full-field optical coherence tomography with a Linnik microscope”, Applied Optics 41 (2002) 805-812]. The arrangements described in each case use a commercial CCD camera as detector. As in an exemplary embodiment of our invention, the camera images are transmitted as data to a computer, and the phase-sensitive demodulation of these data is carried out by numerical analysis with the aid of the computer. The method described in the two publications mentioned is restricted to the separation of constant background light and linear interference signals which are generated by illumination with a light source. In the arrangements described, two light beams having a constant intensity but a modulated phase shift are caused to interfere. The measurement of two-dimensional height profiles, e.g. of surfaces with a patterning of the order of magnitude of micrometers or nanometers, is described as an application of this method. The mathematical method of the Fourier series expansion of a time signal is used in the two publications; only the phase angle of the first harmonic coefficient, which is a measure of the distance between the sample point (measurement location) and the detector, is explicitly used.
By contrast, the present invention does not relate to the measurement of height profiles, distance measurements, or object identification in the case of modulated illumination. Under the conditions of the application of our invention, the position of the measurement location is known very precisely. Moreover, the present invention does not require generation of interference patterns, but is suitable for analyzing these, too, provided that they have nonlinear signal portions. In contrast to the determination—described in the two publications—of signal portions that are correlated linearly with an excitation light from a previously unknown measurement location that is to be determined by this analysis, the present invention is provided for the generation—spatially resolved into one or more spatial coordinates—of images of signals that are correlated nonlinearly with an excitation light and emerge from a well-known measurement location. One possible embodiment of the invention is based on phase-sensitive detection at the second harmonic of the excitation modulation. In this case, the phase angle of the nonlinear signal does not supply any information about the spatial position, but rather about the chemical character, for example. It is thus possible to measure e.g. excitation lifetimes, in contrast to propagation time differences in the case of the publications cited above.